This invention relates to nucleic acid mismatch detection formats.
The ability to detect mutations in coding and non-coding DNA, as well as RNA, is important for the diagnosis of inherited diseases. A gene mutation can be a single nucleotide change or multiple nucleotide changes in a DNA sequence encoding an essential protein. A single nucleotide change or multiple nucleotide changes can result in frame shift mutations, stop codons, or non-conservative amino acid substitutions in a gene, each of which can independently render the encoded protein inactive. However, a gene mutation can be harmless, resulting in a protein product with no detectable change in function (i.e., a harmless gene polymorphism). Mutations in repetitive DNA can also lead to diseases as is the case, for example, in human fragile-X syndrome, spinal and bulbar muscular dystrophy, and myotonic dystrophy.
A mutant nucleic acid that includes a single nucleotide change or multiple nucleotide changes will form one or more base pair mismatches after denaturation and subsequent annealing with the corresponding wild type and complementary nucleic acid. For example, G:A, C:T, C:C, G:G, A:A, T:T, C:A, and G:T represent the eight possible single base pair mismatches which can be found in a nucleic acid heteroduplex, where U is substituted for T when the nucleic acid strand is RNA. Nucleic acid mismatches can form when the two complementary strands of a heteroduplex are derived from DNA or RNA molecules that differ in sequence such that one contains deletions, substitutions, insertions, transpositions, or inversions of sequences compared to the other.
Detection of such mutations provides an important diagnostic tool in areas including cancer diagnosis and prognosis, perinatal screening for inherited diseases, differential diagnosis of diseases not readily detectable by conventional tests (for example, Marfan's syndrome and the fragile X syndrome), and the analysis of genetic polymorphisms (for example, for genetic mapping or identification purposes).